The research objective in this application is to gain further information on the mechanisms of action of the two 4-electron NAD-linked dehydrogenases, UDPG-dehydrogenase and histidinol dehydrogenase. It has been well-established that UDPG-dehydrogenase has two independent active sites that catalyze the overall reaction in 2-electron steps and it is suspected that histidinol dehydrogenase acts in a similar fashion. Evidence has been produced, in both cases, that these two active sites are not located adjacent to each other on the same subunit, but rather adjacent to each other on separate subunits of a dimer pair. The research is designed to produce further evidence in support of this mechanism type and establish how widespread it is in nature. With this in mind UDPG-dehydrogenase from prokaryotic sources will be investigated in a fashion similar to that already done for the beef-liver enzyme. Histidinol dehydrogenase will be investigated to see if it shows typical half-of-the-sites behavior, which it should if the proposed mechanism is correct. Finally, the functional groups connected with each of the active sites of histidinol dehydrogenase will be investigated with a view of determining what they are and devising means to derivatize them with the view of determining the amino acid sequence in the the vicinity of the active site.